Five-antituberculosis Drug-resistance Genes Detection Using Array System

Abstract

Summary Detection of resistance to drugs for Mycobacterium tuberculosis takes about two months from the sample collection using culture-based methods. To test a rapid method for detection of resistance for five antituberculosis drugs using DNA microarray and to examine its potential for clinical use, we employed a DNA microarray for detection of seven mutations genes related to resistance of five kinds of antituberculous drugs using Mycobacterium tuberculosis DNA isolated from sputum. The results of microarray analysis were compared with the results of a standard culture method of Lowenstein-jensen drug sensitivity testing system. DNA microarray analysis showed a high sensitivity (>90%) for all five drugs. Specificity of rifampicin and ethambutol were nearly 90%, however specificity of isoniazid (60%) and kanamycin (67%) were not enough. The amount of Mycobacterium tuberculosis DNA required for microarray analysis corresponded to at least 1-9 Acid-Fast Bacilli per 10 fields by carbolfuchsin staining. DNA microarray analysis appears to be useful for estimation of drug resistances, nevertheless its limitations. To minimize misunderstanding, it is necessary to confirm the number of bacilli in the sputum, and culture method is needed for comparison when use the PCR-based array system.

Rapid and accurate detection of rifampin and isoniazid-resistant Mycobacterium tuberculosis using an oligonucleotide array

“…Microarray analysis of M. tuberculosis has facilitated a range of additional studies to provide important contributions on its mechanisms of drug resistance, host–pathogen interactions, in vitro and in vivo gene expression and functional analysis of particular genes . Previously, several in‐house or commercial arrays were designed for differentiating the M. tuberculosis complex from non‐tuberculous mycobacteria and for rapid detection of MDR and mono‐drug‐resistant M. tuberculosis as well as M. tuberculosis that is resistant to second‐line anti‐TB drugs . The TB‐Biochip oligonucleotide microarray system (Engelhardt Institute of Molecular Biology, Moscow, Russia), evaluated by Caoili et al .…”

Detection of drug resistance in Mycobacterium tuberculosis: Methods, principles and applications

“…The report indicated a high sensitivity (90%) for all five drugs. 67 In this study specificities for RIF and EMB were nearly 90%, but the specificity for INH (60%) and kanamycin (67%) was not satisfactory. More recently, further attempts were started for rapid detection of resistance to fluoroquinolones by microarrays.…”

DNA Microarrays for Pathogen Detection

“…There are a number of commercially available tests for M. tuberculosis available, and they are listed in Table 4.2. Shimizu et al [56] developed a DNA microarray that covered mutations in seven genes (inh, KatG, rpoB, rrs, rpsL, embB) which are responsible for antibiotic resistance against five anti-TB drugs (rifampicin, isoniazid, streptomycin, kanamycin, an ethambutol). The DNA microarray results were compared with standard culture-based sensitivity testing which took 2 months from sample collection.…”

“…The HCII assay is based on hybridization in a solution with a mixture of nonisotopic, single-stranded, full-length RNA probes against 13 high-risk types (HPV 16,18,31,33,35,39,45,51,52,56,58,59,68). The assay detects several high-risk types in one reaction and uses a convenient format.…”